Microbial sensor for new generation cephalosporins based in a protein-engineered beta-lactamase

A protein-engineered beta-lactamase, constructed by site-directed mutagenesis in Escherichia coli (E104M/G238S), and having broadened specificity, was able to degrade cephalosporins of first, second, and third generations. Manipulations of culture conditions allowed an increase in beta-lactamase spe...

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Bibliographic Details
Published in:Applied biochemistry and biotechnology 1998-05, Vol.73 (2/3), p.243-256
Main Authors: Garcia, J.L, Nunez, C.J, Gonzalez, E.G, Osuna, J, Soberon, X, Galindo, E
Format: Article
Language:English
Subjects:
beta-Lactamases - genetics
beta-Lactamases - metabolism
Biological and medical sciences
Biological Sciences
Biosensing Techniques
Biosensors
Biotechnology
Cefamandole
Cefoperazone
Cefotaxime
Cell culture
Cephalosporins
Cephalosporins - analysis
Escherichia coli
Fundamental and applied biological sciences. Psychology
Methods. Procedures. Technologies
Microorganisms
Mutagenesis, Site-Directed
Protein Engineering
Proteins
Site-directed mutagenesis
Various methods and equipments
β Lactamase
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Summary:A protein-engineered beta-lactamase, constructed by site-directed mutagenesis in Escherichia coli (E104M/G238S), and having broadened specificity, was able to degrade cephalosporins of first, second, and third generations. Manipulations of culture conditions allowed an increase in beta-lactamase specific activity by up to twofold. The resultant bacteria were used to construct an immersable whole-cell biosensor for the detection of new-generation cephalosporins. Cells were immobilized on agar membranes, which in turn were attached to the surface of a flat pH electrode, thus constituting a biosensor based on the detection of pH changes. The sensor was able to detect second- and third-generation cephalosporins: cefamandole (0.4.4 mM), cefotaxime (0.4-3.5 mM), and cefoperazone (0.3-1.85 mM). Response times were between 3.5 and 11 min, depending on the kind of cephalosporin tested. The biosensor was stable for at least 7 d, time during which up to 100 tests were performed.
ISSN:0273-2289
1559-0291
DOI:10.1007/BF02785659